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First published online 23 March 2005
doi: 10.1242/dev.01784

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Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

Taro Mito, Isao Sarashina^*, Hongjie Zhang, Akihiro Iwahashi, Haruko Okamoto, Katsuyuki Miyawaki, Yohei Shinmyo, Hideyo Ohuchi and Sumihare Noji

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City 770-8506, Japan

View larger version (57K): [in a new window]   Fig. 1. Alignments of conserved domains of Gb'hb compared with orthologous sequences from other insects. (A) Four central fingers (MF-1 to 4), two C terminal fingers (CF-1 and 2), and A-Box and C-Box of Gryllus bimaculatus (G.b.) hunchback aligned with those of Schistocerca americana (S.a.), Oncopeltus fasciatus (O.f.), Tribolium castaneum (T.c.) and Drosophila melanogaster (D.m.). Arrowheads indicate the structural residues of the putative metal-binding fingers. (B) Schematic structures of various Hunchback proteins, comparing the organization of zinc fingers. Each box shows a zinc finger or C-box. Corresponding zinc fingers of different species are shown in the same shade. Four central fingers (MF-1 to 4) and two C terminal fingers (CF-1 and 2) are conserved in all insects in this figure. The structure of H.t. Hb was predicted from the genomic sequence (Patel et al., 2001; Savage and Shankland, 1996). C.e., Caenorhabditis elegans; H.t., Helobdella triserialis.

View larger version (98K): [in a new window]   Fig. 2. Expression of Gb'hb during embryogenesis. Expression patterns of Gb'hb at 30 (A,B), 32 (C), 36 (D), 40 (E), 42 (F), 48 (G), 52 (H,I) and 72 (J,K) hAEL. The embryo was double-stained with Gb'wg in B (Gb'hb, blue; Gb'wg, brown), E (Gb'hb, brown; Gb'wg, blue) and K (Gb'hb, blue; Gb'wg, brown). (C,D) Stripes of Gb'hb expression in the middle gap domain are indicated by arrowheads. The posterior expression domain (arrowheads in G,H,J) appeared at 48 hAEL (G) and split into two stripes (J). (K) Double staining for Gb'hb (blue) and Gb'wg (brown) indicates that the splitting intense domain of the Gb'hb expression near the posterior end spanned from the A7 stripe to the A9 stripe of Gb'wg. (I) High-magnification view of the boxed area in H, showing spotted expressions. Mn, mandible; Mx, maxilla; Lb, labium; T1-3, thoracic segments 1 to 3; A7-9, abdominal segments 7 to 9. Scale bars: 250 µm in A-H,J; 100 µm in K.

View larger version (88K): [in a new window]   Fig. 3. Effect of Gb'hb RNAi on G. bimaculatus embryos. Anterior towards the left. (A,B) Wild-type embryo in the stage just before hatching. (A) Lateral view. (B) Dorsal view. (C) Wild-type embryo stained for Gb'wg. (D,E) Class III embryo. (D) Lateral view. (E) Dorsal view. (F) Putative class III embryo stained for Gb'wg. In this embryo, segmentation of the T1 to T3 was partially disturbed (arrow). Segmentation defects were frequently observed in Gb'hb RNAi embryos (also shown in H and I). (G,H) Class II embryo. (G) Lateral view. Arrowhead indicates a defective T3 leg. (H) Dorsal view. (I) Putative class II embryo stained for Gb'wg. Arrows in H and I indicate segmentation disturbances. (J-L) Class I embryo. (J) Lateral view. Arrowhead indicates the mandible. (K) Dorsal view. (L) Ventral view. Arrowhead indicates a vestige of a T3 leg. (M) Putative class I embryo stained for Gb'wg. (N-S) Sagittal sections through wild-type and Gb'hb RNAi embryos. (N,Q) Wild type. (O,R) Class II embryo. (P,S) Class I embryo. Each of these pairs was derived from the same specimen. The digestive tract consists of a fore-, mid- and hindgut. (N) In wild type, the pharynx (ph), esophagus (es), crop (cr) and proventriculus (pv) are observed in the foregut. The midgut contains yolk granules. (O) In class II embryos, the fore- and hindgut were compressed and the midgut was relatively shifted towards the anterior. (P) In more severe cases, a sac-like midgut was observed, and the fore- and hindgut were further compressed. (Q) The musculature in the abdominal segments shows a reiterated pattern (arrowheads). (R,S) In Gb'hb RNAi embryos, musculature was observed in each segment, although the reiterated pattern was not orderly. Arrows in P and R indicate a vestige of the T3 leg. an, antenna; cr, crop; es, esophagus; fg, foregut; ph, pharynx; mo, mouth; pv, proventriculus; mg, midgut; hg, hindgut; T, thorax; Mn, mandible; Mx, maxilla; Lb, labium; T1-3, thoracic segments 1 to 3. Scale bars: in A, 400 µm for A,B,D,E,G,H,J,K,L; in N, 400 µm for N-S; 200 µm in C,F,I,M.

View larger version (76K): [in a new window]   Fig. 4. Expression patterns of Hox genes in embryos depleted of Gb'hb by RNAi. (A) Wild-type embryo stained for Gb'abdA. Gb'abdA is expressed in the posterior compartment of A1 and the remaining abdominal segments. (B-D) Putative class III-I embryos stained for Gb'abdA. Ectopic expression of Gb'abdA in gnathal and thoracic regions was observed in B and C, suggesting that the strong phenotype (D) consists of transformed gnathal and thoracic segments and a shortened abdomen. (E) Wild-type embryo stained for Gb'Scr. Gb'Scr is strongly expressed in the labial and T1 segments. Expression in the labial segment is more intense than in the T1 segment. (F-H) Putative class III-I embryos stained for Gb'Scr. Gb'Scr expression in the labial and T1 segments was gradually reduced as the phenotype became more severe. (I) Wild-type embryo stained for Gb'Antp. The anterior boundary of the Gb'Antp expression is the T1 segment. (J-L) Putative class III-I embryos stained for Gb'Antp. The anterior boundary of the Gb'Antp expression was shifted to the labial segment in all phenotype classes. The strong midline expression in the anterior region is an abdomen-like pattern and probably ectopically induced. (M) Wild-type embryo stained for Gb'Ubx. Gb'Ubx is strongly expressed in the T3 legs and A1 prolegs, and weakly expressed in the abdominal segments. In the abdomen, the intensity of expression follows a gradient from anterior to posterior. (N-P) Putative class III-I embryos stained for Gb'Ubx. (N) Expression patterns of Gb'Ubx indicates that the leg most resistant to RNAi (arrowhead) was T3. Strong expression of Gb'Ubx in the A1 prolegs was also detected in this class of embryos (arrow). Scale bar: 250 µm.

View larger version (24K): [in a new window]   Fig. 5. Alignment of Gb'Kr with orthologous sequences of Drosophila melanogaster (Dm) and Tribolium castaneum (Tc). The labeled brackets indicate zinc fingers (type 1 to 4).

View larger version (108K): [in a new window]   Fig. 6. In situ detection of segmentation and Hox genes in early-stage embryos depleted of Gb'hb by RNAi. (A-C) Expression patterns of Gb'Kr in wild-type (A) and Gb'hb RNAi embryos (B,C) at 40-42 hAEL. Gb'Kr is expressed in the gnathal and thoracic regions at this stage in wild-type embryos. Expression in this domain was reduced by Gb'hb depletion and, in severe cases, almost eliminated. The embryo shown in C was affected more severely than that shown in B. Gb'hb RNAi embryos are shorter and flatter than the wild type. (D-F) Expression patterns of Gb'eve in wild-type (D) and Gb'hb RNAi embryos (E,F) at 38-40 hAEL. In wild-type embryos at this stage, Gb'eve is expressed as five clearly resolved stripes and an additional one or two stripes are resolving from the posterior broad domain. (E) In Gb'hb RNAi embryos, the number of stripes was reduced to less than five. (F) In more severe cases, only three stripes were observed. (G-I) Expression patterns of Gb'wg in wild-type (G) and Gb'hb RNAi embryos (H,I) at 45-47 hAEL. (G) At this stage of wild type, seven Gb'wg stripes are observed in the gnathal and thoracic regions. (H) In Gb'hb RNAi embryos, the stripes in the thoracic region were affected in that they had an irregular shape and the number of stripes was reduced from three to two. (I) In more severe cases, the labial stripe was also disturbed. (J,K) Expression patterns of Gb'Antp (brackets) in wild type (J) and a Gb'hb RNAi embryo (K) at 38-40 hAEL. (K) The Gb'Antp domain was severely reduced in Gb'hb RNAi embryos. (L,M) Expression patterns of Gb'abdA in wild type (L) and a Gb'hb RNAi embryo (M) at 45-47 hAEL. (L) In wild type, the Gb'abdA expression in the abdominal region begins close to this stage. (M) In Gb'hb RNAi embryos, the ectopic Gb'abdA expression was observed in the gnathal and thoracic regions. Scale bar: 250 µm.

View larger version (25K): [in a new window]   Fig. 7. Schematic drawing of the effects of Gb'hb RNAi during embryogenesis. All embryos are shown from a ventral view. (A) Wild-type and Gb'hb RNAi embryos at early germband stage (38-42 hAEL). In Gb'hb RNAi, early germbands are shortened and flattened. Reduction of Gb'Antp expression domain indicates that the prospective thoracic region is reduced in Gb'hb RNAi embryos. (B) Wild-type and Gb'hb RNAi embryos at the beginning of germband elongation. Vertical lines on the embryos represent the anterior segmental stripes of Gb'wg. The stripes affected by Gb'hb RNAi segments are indicated by red arrowheads in the wild-type embryo, with the deleted region (posterior T1 to anterior T2) shown by a red bracket. In Gb'hb RNAi, the Gb'wg stripes indicated by dotted lines are not eliminated but irregularly shaped. Gb'abdA is expressed in a limited domain in the posterior region of the wild-type embryo, while its ectopic expression is induced in the anterior region of the Gb'hb RNAi embryos. Accordingly, the segment identities of Gb'hb RNAi embryos change in the prospective mandible to T3 segments (labeled by parenthesized abbreviations). (C) Wild-type and Gb'hb RNAi embryos at stage 9. The Gb'hb RNAi phenotype is a combination of the transformation of the gnathal and thoracic regions (the maxillary to T3 segments) towards the abdominal identity and the reduction of the number of segments in the abdomen. The vestiges of T3 legs (arrowhead) are observed in most RNAi embryos. The phenotype would also represent the deletion of one segment, as inferred from the modification of Gb'wg stripe pattern resulting from the fusion of the T1 and T2 segments, though it was not obvious from the inspection of embryos in later stages because of segmentation disturbances. (D) Comparison of the hb functions in Gryllus and Drosophila. Red bars indicate deletions in the affected regions, while yellow bars indicate transformation into abdominal identity (or more posterior identity in the A1 segment of Drosophila). For Drosophila, the case of the class V alleles (Lehmann and Nüsslein-Volhard, 1987) is shown. In Gryllus, only three abdominal segments (dark blue arrow) are formed in severe cases of Gb'hb RNAi depletion, probably owing to the defect in the posterior growth accompanied by segmentation. (E) Models for regulatory networks of hb and other segment-patterning genes in the anterior region of Drosophila and Gryllus. In Drosophila, bcd activates hb and Kr in the anterior region of the embryo. hb and bcd activate eve (stripe 2), while Kr represses it. In Gryllus, hb is activated by cad and regulates the Kr and eve expression in the thoracic region, directly or indirectly. Gryllus hb and probably Kr repress abdA as in Drosophila (see text).

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